Project Summary Breast cancer is the second-leading cause of cancer-related deaths in women. Over two thirds of breast cancers are estrogen receptor (ER) and progesterone receptor (PR) positive (termed luminal), and account for the majority of breast cancer deaths. Following endocrine treatments, residual luminal breast cancer cells can become dormant and pose a risk of recurrence that can last up to 20 years. The existence of a rare population of cancer stem cells (CSCs) that are more quiescent, self-renewing, invasive, drug resistant, and tumor initiating than non-CSCs may explain the prolonged risk of recurrence in luminal breast cancer. Our laboratory has previously identified the intermediate filament protein cytokeratin 5 (CK5) as a marker of luminal breast CSCs. These cells are relatively rare in luminal breast cancers but can be expanded by progesterone or estrogen deprivation. CK5 knockdown, knockout, and overexpression in luminal breast cancer cell lines has indicated that CK5 is both necessary and sufficient, respectively, for mammosphere formation, a measurement of cancer cell self-renewal. This suggests that CK5 may have a functional role in maintaining a luminal breast CSC population, but how it accomplishes this is still unknown. Although cytokeratins are largely regarded as structural proteins that protect epithelial cells from stress, recent studies have identified that cytokeratins affect cell signaling through protein-protein interactions. We performed an unbiased immunoprecipitation (IP)-mass spectrometry screen to identify novel CK5 interacting proteins in luminal breast cancer cells. We identified ?- catenin, the key transcription factor of the Wnt signaling pathway and essential component of adherens junctions, as an interactor of CK5 and confirmed this interaction in luminal and basal breast cancer cell lines and a PDX model. We found that in addition to increasing mammosphere formation and CK5 expression, progesterone was capable of increasing ?-catenin transcriptional activity, which was abrogated by CK5 CRISPR knockout. Furthermore, both CK5 overexpression and progesterone treatment caused loss of membrane ?-catenin which could have implications in the invasive potential of these cells. Several members of the 14-3-3 family of scaffolding proteins were identified by IP-MS and confirmed by co-IP. These proteins are known to interact with ?-catenin. Therefore, this proposal tests the hypothesis that progesterone induced CK5+ CSCs rely on altered ?-catenin dynamics mediated by 14-3-3 proteins to promote luminal breast tumor progression. The specific aims of this proposal are to 1) evaluate the dependence of progesterone induced CSCs on CK5 and ?-catenin dynamics, and 2) determine if the CK5/14-3-3 interaction is necessary for altered ?-catenin dynamics. These studies will define novel protein interactions that contribute to luminal breast CSC maintenance, which could ultimately lead to development of new strategies to prevent recurrence.